Influence of a sulfhydryl cross-link across the allosteric-site interface of E. coli phosphofructokinase

Protein Science : a Publication of the Protein Society
J L JohnsonG D Reinhart

Abstract

To assess the role of quaternary stability on the properties of Escherichia coli phosphofructokinase (PFK), a disulfide bond has been introduced across the subunit interface containing the allosteric binding sites in E. coli phosphofructokinase by changing N288 to cysteine. N288 is located in close proximity to the equivalent residue on an adjacent subunit. Although SDS-PAGE of oxidized N288C indicates monomeric protein, blocking the six native cysteine residues with N-ethyl maleimide (NEM) reveals dimers of N288C on non-native gels. Subsequent addition of dithiothreitol (DTT) to NEM-labeled N288C regenerates the monomer on SDS-PAGE, reflecting the reversibility of intersubunit disulfide bond formation. KSCN-induced hybrid formation between N288C and the charged-tagged mutant E195,199K exhibits full monomer-monomer exchange only upon DTT addition, providing a novel assessment of disulfide bond formation without NEM treatment. N288C also exhibits a diminished tendency toward nonspecific aggregation under denaturing conditions, a phenomenon associated with monomer formation in PFK. Pressure-induced dissociation and urea denaturation studies further indicate that oxidized N288C exhibits increased quaternary stability along both in...Continue Reading

References

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